1. Field of the Invention
The invention is generally in the field of molecular biology as related to the control of programmed cell death. The invention also relates to transgenic non-human animals comprising a disrupted Ich-3 (Caspase-11) gene. This invention further relates to methods of making and using the transgenic animals.
2. Related Art
Programmed Cell Death
Apoptosis, also referred to as programmed cell death or regulated cell death, is a process by which organisms eliminate unwanted cells. Such cell death occurs as a normal aspect of animal development as well as in tissue homeostasis and during aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 26:59-86 (1950); Ellis et al., Dev. 112:591-603 (1991); Vaux et al., Cell 76:777-779 (1994)). Programmed cell death can also act to regulate cell number, to facilitate morphogenesis, to remove harmful or otherwise abnormal cells and to eliminate cells that have already performed their function. Additionally, programmed cell death is believed to occur in response to various physiological stresses such as hypoxia or ischemia. The morphological characteristics of apoptosis include plasma membrane blebbing, condensation of nucleoplasm and cytoplasm and degradation of chromosomal DNA at inter-nucleosomal intervals. (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9-34).
Apoptosis is achieved through an endogenous mechanism of cellular suicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9-34) and occurs when a cell activates its internally encoded suicide program as a result of either internal or external signals. The suicide program is executed through the activation of a carefully regulated genetic program (Wylie, A. H., et al., Int. Rev. Cyt. 68: 251 (1980); Ellis, R. E., et al., Ann. Rev. Cell Bio. 7: 663 (1991); Yuan, Y. Curr. Op. Cell. Biol. 7:211-214 (1995)).
In many cases, gene expression appears to be required, since cell death can be prevented by inhibitors of RNA or protein synthesis (Cohen et al., J. Immunol. 32:38-42 (1984); Stanisic et al., Invest. Urol. 16:19-22 (1978); Martin et al., J. Cell Biol. 106:829-844 (1988). A genetic pathway of programmed cell death was first identified in the nematode C. elegans (Ellis, R. E., et al., Annu. Rev. Cell Biol. 7:663-698 (1991)). In this pathway, the function of two genes, ced-3 and ced-4, are required for cells to undergo programmed cell death Genetic mosaic analysis indicated that both ced-3 and ced-4 most likely act in dying cells to induce cell death; thus, they are essential parts of intracellular machinery involved in execution of cell death (Yuan & Horvitz, Dev. Biol. 138:33-41 (1990)). Furthermore, in C. elegans, the products of ced-3 and ced-4 genes carry out the program of cellular suicide (Yuan & Horvitz, Dev. Bio. 138: 33 (1990)).
Amino acid sequence of CED-3 protein is homologous to mammalian interleukin-1.beta. converting enzyme (ICE) with 28% amino acid identity (Yuan et al., Cell 75:641-652 (1993)). The C terminal half of the CED-3 is more homologous to ICE (43% identity), which includes the active pentapeptide QACRG present in all members of the ICE/CED-3 family.
Interleukin-1-.beta. Converting Enzyme (ICE) Family
The interleukin-1.beta. converting enzyme (ICE) family is a growing family of cysteine proteases involved in cytokine maturation and apoptosis (Yuan, J., Curr. Opin. in Cell Biology 7:211-214 (1995)). ICE is a cytoplasmic cysteine protease responsible for proteolytically processing pro-interleukin-1.beta. (31 kDa) into active form (17 kDa) (Thornberry, N. A., Nature 356:768-774 (1992), Cerretti, D. P., et al., Science 256:97-100 (1992)). ICE is synthesized as a precursor of 45 kDa which is proteolytically cleaved during activation to generate two subunits of 22 kDa p20) and 10 kDa (p10) (Thornberry, N. A., et al., Nature 356:768-774 (1992)). X-ray crystallography analysis of three dimensional structure of ICE showed that ICE is a homodimer of activated ICE p20 and p10 subunits (Wilson, K. P., et al., Nature 370:270-275 (1994); Walker, N. P. C., et al., Cell 78:343-352 (1994)). Activated ICE can cleave the inactive ICE precursor; however, in vitro synthesized ICE precursor cannot cleave itself (Thornberry, N. A., et al., Nature 356:768-774 (1992)), suggesting that ICE may need to be activated by another protease in vivo.
The amino acid sequence of ICE shares 29% identity with C. elegans cell death gene product Ced-3 (Yuan et al., Cell 75:641-752 (1993)) which suggests that ICE may play a role in controlling mammalian apoptosis.. Expression of Ice in a number of mammalian cell lines induces apoptosis (Miura et al., Cell 75:653-660 (1993); Wang et al., Cell 87:739-750 (1994)). Microinjection of an expression vector of crmA, a cowpox virus gene encoding a serpin that is a specific inhibitor of ICE, prevents not only death of neurons from dorsal root ganglia induced by trophic factor deprivation but also the death of ciliary ganglia (Gagliardini et al., Science 263:826-828 (1994); Li et al., Cell 80:401-411 (1995); Allsopp et al., Cell 73:295-307, (1993)). Expression of crmA can also suppress apoptosis induced by TNF-.alpha. and Fas (Enari et al., Nature 375:78-81 (1995); Los et al., Nature 375:81-83 (1995); Kuide et al., Science 267:2000-2002 (1995); Miura et al., Proc. Natl. Acad. Sci. U.S.A. 92:8318-8322 (1995)). These experiments suggest that the members of the ICE family play important roles in controlling mammalian apoptosis. These results did not indicate, however, which member of the ICE family is critical for cell death since CrmA may cross-inhibit other members of the ICE family.
The mammalian ICE/CED-3 family now includes eight members: ICE, TX/ICE.sub.rel II/ICH-2, ICE.sub.rel III, ICH-1/NEDD2, CPP32/Yama/Apopain, MCH2, MCH-3/ICE-LAP3/MCH-2 and ICH-3 (Kumar et al., Genes Dev. 8:1613-1626 (1994); Fernandes-Alnemri, et al., J. Biol. Chem. 269:30761-30764 (1994); Fernandez-Alnemri et al, Cancer Res. 55:2737-2742 (1995); Fernandes-Alnemri et al., Cancer Res. 55:6045-6052 (1996); Wang et al., Cell 78:739-750 (1994); Faucheu, et al, EMBO J. 14:1914-1922(1995); Tewari & Dixit, J. Biol. Chem. 270:3255-3260 (1995); Kamens et al., J. Biol Chem. 270:15250-15256 (1995); Munday, N. A., et al., J. Biol. Chem. 270:15870-15876 (1995); Duan, H. J., et al., J. Biol. Chem. 271:1621-1625 (1996); Lippke, J. A., et al., J. Biol. Chem. 271:1825-1828 (1996)). Since ICH-3 is most homologous to TX, it may be the mouse version of human TX. This cannot be concluded at the moment, however, because TX has been shown to cleave pro-ICE (Faucheu, et al., EMBO J. 14:1914-1922 (1995)) whereas ICH-3 has not been shown to cleave pro-ICE in a similar assay (data not shown). The current designation of ICH-3 is Caspase-11.
Overexpression of Nedd-2/Ich-1.sub.L induces cell death very effectively (Kumar et al., Genes Dev. 8:1613-1626 (1994); Wang et al., Cell 87:739-750 (1994)). Expression of CPP32/Yama in full length cDNA induces apoptosis of insect Sf9 cells but not that of mammalian cells (Fernandes-Alnemri et al., J Biol. Chem. 269:30761-30764 (1994); E. S. Alnemri, personal communication). Recombinant CPP32/Yama is inactive and cleavage of CPP32/Yama by ICE in vitro activates the precursor (Tewari et al., Cell 81:801-809 (1995b)), suggesting that in vivo CPP32(Yama may be activated by another protease to induce apoptosis. Expression of MCH2.alpha. also induces apoptosis of insect Sf9 cells but not that of mammalian cells (Fernandes-Alnemri et al., Cancer Res. 55:2737-2742 (1995)). Thus, the members of the ICE family can be classified into 2 classes: those that when overexpressed in mammalian cells can induces apoptosis (e.g. Ice and Ich-1) and those that when overexpressed in mammalian cells cannot induce apoptosis (e.g. CPP32 and Mch-2). These experimental evidence suggest that in vivo members of the ICE family may be arranged in proteases cascades and certain members of the ICE family may activate other members of the ICE family.
The control of apoptosis in mammals is much more complex than that in C. elegans where function of one ced-3 gene controls all programmed cell death (Ellis & Horvitz, Cell 44:817-829 (1986)). In contrast to C. elegans, multiple proteases may be involved in regulation of programmed cell death (apoptosis) in mammals. This hypothesis is supported by many in vitro studies. For instance, peptide inhibitors of ICE such as YVAD-cmk inhibit Fas induced apoptosis but requires much higher doses than that for inhibiting ICE (Enari et al., Nature 375:78-81 (1995)), suggesting that inhibition of additional ICE-like protease(s) is required for complete inhibition of Fas induced apoptosis. Similarly, Ac-DEVD-CHO, a peptide inhibitor of CPP32/Yama/Apopain, inhibits poly(ADP-ribose) polymerase (PARP) cleavage at a dose of 1 nM but requires 1 .mu.M to cause 50% inhibition of apoptosis in an cell-free system (Nicholson, D. W., et al., Nature 376:37-43 (1995)), suggesting that inhibition of protease(s) other than CPP32/Yama/Apopain is required for complete inhibition of apoptosis in this system. Furthermore, inhibitors that are known not to have effects or have little effects on ICE like cysteine proteases such as cysteine protease inhibitors trans-epoxysucciniyl-L-leucylamido-(4-guanidino) butane (E64) and leupeptin, calpain inhibitors I and II, and serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluroride, were found to inhibit apoptosis induced by T cell receptor binding-triggered apoptosis (Sarin et al., J. Exp. Med. 178:1693-1700 (1993)), suggesting that not only cysteine proteases but also serine proteases may play important roles in mammalian cell apoptosis.
Cytotoxic T lymphocytes (CTL) are important players in host cell-mediated immunity (reviewed by Henkart & Sitkovsky, Curr. Opin. in Immun. 5:404-410 (1994)). Granzyme B(GraB) is a serine protease Granzyme B is a serine protease required for the cytotoxic activity of lymphocytes (Shi et al., J. Exp. Med. 176:1521-1529(1992)). It also plays a major role in apoptosis induced by CTLs since mice that are deficient for GraB generated by gene targeting technique are severely defective in CTL induced apoptosis (Heusel, J. W., et al., Cell 76:977-987 (1994)). GraB can induce apoptosis of many if not all cell types in the presence of pore forming protein perforin (Shi et al., J. Exp. Med. 175:553-566 (1992) & Shi et al., J. Exp. Med. 176:1521-1529 (1992)).
Recent work showed that apoptosis of embryonic fibroblasts induced by granzyme B is mediated through ICE (Shi et al., Proc. Natl. Acad. Sci. In Press (1996)) Apoptosis induced by granzyme B and perforin can be inhibited by inhibitors of the ICE family, including CrmA, ICH-1.sub.S and a mutant ICE (Shi et al., Submitted (1996)). Most significantly, embryonic fibroblasts from Ice deficient mice are resistant to granzyme B/perforin induced apoptosis, suggesting that ICE itself is required for cytoxicity of granzyme B/perforin in at least certain cell types (Shi et al, Submitted (1996)). Granzyme B does not, however, cleave and activate ICE precursor directly (Darmon, A. J., et al., J. Biol. Chem. 269:32043-32046 (1994)), suggesting that there are intermediate steps of regulation between granzyme B and ICE.
A recent report showed that CPP32, a member of the ICE family, is activated by cytotoxic T-cell-derived GraB, suggesting that CPP32 is important for CTL killing (Darmon, A. J., et al., Nature 377:446-448 (1995)). CPP32, however, cannot be the only ICE family activated by CTL since CrmA is a very poor inhibitor of CPP32 (Nicholson, D. W., et al, Nature 376:37-43 (1995)). Tewari et al., Chem. 270:22605-22708 (1995) showed that expression of crmA completely blocks the Ca.sup.2+ -independent component of CTL-killing (i.e. Fas-mediated); if CPP32 were the only ICE family member responsible for CTL cytotoxicity, expression of crmA should not suppress CTL killing. It is predicted that there are additional members of the ICE family which play an important role in CTL induced apoptosis. The amino acid sequence of GraB is not homologous with ICE; however, GraB and ICE share many enzymatic similarities. Like ICE, GraB requires Asp at P1 position for cleavage. Inhibitors of ICE or the ICE family, CrmA, ICH-1.sub.s and a mutant ICE are effective inhibitors of GraB/perforin induced apoptosis (Shi et al., Submitted (1996)). Embryonic fibroblasts that are deficient in ICE from Ice-/- mice are resistant to GraB/perforin induced apoptosis (Shi et al, Submitted (1996)), suggesting that ICE is critical for GraB/perforin induced apoptosis in at least certain cell types. ICE itself cannot be directly cleaved by GraB (Darmon, A. J., et al., J. Biol. Chem. 269:32043-32046 (1994)) and thus, although ICE is required for GraB/perforin induced apoptosis in certain cells, GraB does not activate ICE directly. One possibility is that GraB activates another ICE family member which may then directly or indirectly activate ICE and the activator of ICE can be inhibited by CrmA.
Transgenic Animals
With so many members in the ICE/CED-3 family, it is important to determine the ICE/CED-3 family member's functions individually. Transgenic mice are an ideal model for accomplishing this by generating mutations in the genes of interest, resulting in "knock-out" mice. Using such models, it has already been shown that mice deficient in Ice develop normally but are resistant to endotoxic shock induced by lipopolysaccharide (LPS). This can be attributed to their defect in production of mature IL-1.beta. (Li et al., Cell 80:401-411 (1995); Kuida et al., Science 267:2000-2003 (1995)). Furthermore, Ice deficient thymocytes undergo apoptosis normally when stimulated with dexamethasone and .gamma.-irradiation but are resistant to Fas induced apoptosis (Kuida et al., Science 267:2000-2003 (1995)), suggesting that ICE is required for Fas but not dexamethasone and .gamma.-irradiation induced apoptosis in thymocytes. Ice may be involved, however, in .gamma.-irradiation induced cell death in concanavalin A (conA)-stimulated splenocytes (Tamura et al., Nature 376:596-599 (1995)). Expression of Ice is induced in splenocytes stimulated by conA and induction of Ice expression enhances the susceptibility of mitogen activated T cells to cell death induced by .gamma.-irradiation and DNA-damaging chemotherapeutic agents such as adriamycin or etoposide induced cell death.
Generation of mutant mice by gene targeting technique and ultimately, making crosses all of potential candidate genes, should provide vital information about the genetic and biochemical pathways of apoptosis. Over the last several years, transgenic animals containing specific genetic defects, e.g., resulting in the development of, or predisposition to, various disease states, have been made. These transgenic animals can be useful in characterizing the effect of such a defect on the organism as a whole, and developing pharmacological treatments for these defects.
The relevant techniques whereby foreign DNA sequences can be introduced into the mammalian germ line have been developed in nice. See Manipulating the Mouse Embryo (Hogan et al., eds., 2d ed., Cold Spring Harbor Press, 1994) (ISBN 0-87969-384-3). At present, one route of introducing foreign DNA into a germ line entails the direct microinjection of a few hundred linear DNA molecules into a pronucleus of a fertilized one-cell egg. Microinjected eggs may then subsequently be transferred into the oviducts of pseudo-pregnant foster mothers and allowed to develop. It has been reported by Brinster et al. (1985), that about 25% of the mice that develop inherit one or more copies of the microinjected DNA.
More specifically, "knock-out" mice, a specific type of transgenic animal, are obtained by first making mutant ES cells. Chimeric mice are then made by injecting ES cells into blastocytes and the chimera are bred to obtain the germline transmitted mutation.
In addition to transgenic mice, other transgenic animals have been made. For example, transgenic domestic livestock have also been made, such as pigs, sheep, and cattle. Once integrated into the germ line, the foreign DNA may be expressed in the tissue of choice at high levels to produce a functional protein. The resulting animal exhibits the desired phenotypic property resulting from the production of the functional protein.
In light of the various biological roles of apoptosis, there exists a need in the art to develop transgenic animals, e.g., transgenic mice, wherein genes involved in apoptosis have been modified. There also exists a need in the art to develop methods to test compounds directed to modifying the apoptotic condition using these transgenic animals. A further need in the art is to develop treatments for various pathological states in which apoptosis has been found to occur.